Fibrinolysk (1993) 7, Suppl 1 : 17-23 g, 1993 ,+x~gman Group UK Ltd

Fibrinolytic Tests and Extravascular Proteolysis

Urokinase and Urokinase Receptor: a Paracrine/Autocrine System Regulating Cell Migration and Invasiveness

Francesco Blasi

Dipartimento di Genetica e Biologia dei Microrganismi, University of Milano, and DIBIT, H. S. Rafiaele, via Olgettina 58,20132 Milano, Italy

Extracellular proteolysis is required in normal and patho- logical forms of cellular invasiveness (i.e. in embryonic development, during inflammatory responses, cancer metastasis and spreading). Urokinase (u-PA) and its re- ceptor (u-PAR) are essential components in the regula- tion of extracellular proteolysis and thus regulate the cell migration machinery, providing an inducible, transient and localized cell surface proteolytic activity. The uro- kinase receptor allows a continuous regulation of the proteolytic activity at cell contacts, utilizing the different localization of urokinase and its inhibitors. The receptor, in fact, in addition to focusing the enzymatic activity at focal and cell-cell contacts, also regulates it by intema- lizing and degrading only the inhibited form of uroki- nase. u-PAR’-~ is a 281 aminoacids long cell surface receptor which is anchored to the membrane through a glycophospholipid moiety. It is synthesized as a 313 residues protein, the carboxyterminal32 being processed in the process of maturation and GPI anchoring.4’ 5 u- PAR is made up of three different domains, the amino terminal one being endowed with the ligand-biding ca- pacity. The ligand-binding can be both pro-u-PA as well as u-PA, which utilize the growth-factor domain (residues 20-32) for u-PAR recognition.1’6 Receptor bound pro-u-PA is activated to u-PA at a much higher rate4 than in solution and therefore the cell surface is the preferential site and parameter regulating extracellular proteolysis and cell migration.

Receptor-bound u-PA is sensitive to the specific plas- minogen activator inhibitors, PAI-1, PAT-2 and protease nexins. This is a very important regulatory step since it not only blocks the production of plasmin, but also modifies the areas of the cell surface that are in a prote- olytically active state. Indeed, while pro-u-PA and ac- tive u-PA when bound to u-PAR remain in an active state on the cell surface, the u-PA: inhibitors complexes are internalized and degraded.7’ ’ The inhibitors there-

fore cause a change of state of u-PAR turning it from an anchorage-protein into an internalizing receptor.

Internalized receptor releases the ligands to the lyso- somes and recycles back to surface. In this way, the proteolytically active areas of the cell surface can be continuously monitored for their activity and their loca- tion modified.g The cell can thus coordinate its migra- tion efforts with a step-wise modification of the proteolytic activity-map of the cell surface. Intemaliza- tion of u-PAR-bound u-PAPA1 complexes, however, re- quires the participation of an additional, transmembrane protein. The a-2 macroglobulin receptor (also called LRP for Lipoprotein Receptor Related Protein), in fact, drives the internalization of the u-PA:PAI-1 complex. LRP, however, does not bind u-PA:PAI directly and therefore it probably recognizes an epitope in U-PA:PAI complex which is evidenced by the binding to u-PAR.9

The urokinase cycle (synthesis, secretion, receptor- binding, activation, inhibition by PAI and internaliza- tion-degradation) can be supported by one individual cell (autocrine2) or by two or more cells. In the latter case, complementation and synergism of urokinase and its re- ceptor are found. This can be demonstrated both in model systems and in, at least some, human tumours. Mouse cells can be transfected with either the human u- PA or the human u-PAR gene, and the synergistic need for the two molecules demonstrated in, for example, ex- tracellular m$rix degradation and invasion of basement membranes. In human colon adenocarcinomas, u-PA appears to be produced by stromal fibroblastic cells while u-PAR is present in large amounts in tumour cells.

The possibility of using this information to diagnose or treat the metastatic aspects of human cancers is cur- rently under intense investigation. In particular, soluble forms of u-PAR can be found in the plasma of patients of ovarian adenocarcinomas and may be used as markers of active invasive activity. Finally, soluble forms of u- PAR can be prepared in the laboratory and may serve as master-tools in the search for agents that block the spreading of cancer cells by blocking the u-PA:u-PAR interaction.

References

1. Stoppclli MI’, Corti A, Sofflentini A, Cassani G, Blasi F, Associan RK. Differentiation-enhanced binding of the amino terminal fragment of human umkinase plasminogen activator to a specific receptor on U937 monocytes. Pmc Nat1 Acad Sci USA 1985; 82: 4939-4943.

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18 Elbrinolytic Tests and E!xtravascular Proteolysis

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Stoppelli MP, Tacchetti C, Cuhellis MV et al. Autocrine saturation of pro-urokinase receptors on human A431 cells. Cell 1986; 45: 675-684. Roldan AL, Cubellis M V, Masucci MT, et al. Cloning and expression of the receptor for human urokinase-plasminogen activator, a central molecule in cell-surface plasmin-dependent protcolysis. EMBO (Eur Mol Biol Org) J 1990.9: 467-474. Plough M, Renne E, Behtendt N, Jensen AL, Blasi F, Dane K. Cellular receptor for urokinase plasminogen activator: carhoxy-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem 1991; 266: 1926-1933. Mller LB, Ploug M, Blasi F. Structural requirements for glycosyl-phosphatidylinositol anchor attachment in the cellular receptor for umkinase plasminogen activator. Eur J Biochem 1992; 208: 493-500. Appella E, Robinson EA, Ulhich SJ, et al. The receptor binding sequence of umkinase. A biological function for the growth factor module of proteases. J Biol Chem 1987; 262: 4437-4440. Cuhellis MV, Wun T-C, Blasi F. Receptor-mediated internalization and degradation of utokinase-PAl-1 complex in human U937 cells. EMBO J. 1990,9: 1079-1085. Olson D, P6ll&ten J, Renne E, et al. Internalization of the urokinase:Plasminogen Activator Inhibitor type 1 complex is mediated by the tnokinase receptor. J Biol Chem 1992; 267: 9129-9133. Blasi F. Urokinase and urokinase receptor: a paracrine/autocrine system regulating cell migration and invasiveness. BioEssays 1993; 15: 105-111. Ossowski L, Chmie G, Masucci M-T, Blasi F. In vivo patacrine interaction between urokinase and its receptor: effect on tumour cell invasion. J Cell Biol 1991; 115: 1107-l 112.

The Role of Fibrinolysis in the Cross-talks Among Vessel Wall Components: the Vitronectin-PAI- Axis

Klaus T. Preissner’, Christine Kost’, Sylvia Rosenblatt’, Hetty de Bee?, Hans-Peter Hammes3, and Hans Pannekoek4

1 Haemostasis Research Unit, Max-Planck-Institutfr physiologische und klinische Forschung, Kerckhoff- Klinik, D-6350 Bad Nauheim,Germany 2Dept. of Haematology, University Hospital, NL-3508 Utrecht, The Netherlands 3Medizinische Polyklinik, University Hospital, D-6300 Giessen, Germany 4Dept. of Molecular Biology, CLB, NL-1066 Amsterdam, The Netherlands

The humoral defense mechanisms in mammalian organ- isms involve reaction cascades such as the contact, blood coagulation, fibrinolysis, and complement systems, whose regulatory functions are localized and expressed at distinct stationary phases, such as cell surfaces, fibril- lar arrays or basement membranes. At any stage, humo-

ral as well as matrix-associated inhibitors provide a con- trol of pro-coagulant and pro-fibrinolytic enzymes, so that clot formation as well as ongoing dissolution of the fibrin clot finally becomes terminated and the patency of the vessel is regained. While these processes appear to be a traditionally recognized concept, basic models of in- teraction between blood vessel components including extracellular matrix (ECM) and humoral factors from the circulation are largely not well understood.

The characteristics of vessel wall basement mem- branes and ECM are mainly determined by the type of structural elements, primarily various collagens, laminin, tibronectin, proteoglycans and others. The association of additional adhesive glycoproteins - such as vitronec- tin (VN) and other morpho-regulatory factors - with the ECM of normal vessels, will endow these sites with new functional properties.’ ) 2 In particular, components of the plasminogen activation system (plasminogen activa- tor inhibitor type-l lPAI-11. plasminogen and others) bind to VN and remain fixed within the matrix compart- ment. In addition, hydrolytic enzymes (serine proteases, glycanases, phospholipases) and growth regulatory ele- ments are orchestrated to serve important pericellular functions which are relevant for growth control and the homeostasis of the vascular system. Modification of cel- lular adhesion mechanisms by pericellular proteolysis is required for normal cell migration and cell invasion necessary for inflammatory or wound healing processes; dysregulation of these events may result in connective tissue damage, tumour cell invasion and metastasis.3 Major control of plasminogen activation at these sites is achieved by PAI-1, which is present in platelets and pro- duced by a variety of cell lines, and which acts predomi- nantly as a fast inhibitor of both tissue-type (t-PA) as well as urokinase-type (u-PA) plasminogen activators.4 The active form, which is associated with a high Mr PAI- protein fraction relates to the existence of VN- PAI- complexes.5 PAL1 deposited in the subendothe- hum appears to be stabilized by binding to matrix- associated VN. Also, release of VN-PAL1 complexes upon platelet stimulation results in the formation of a platelet plug containing PAL1 in its active form. Poss- ibly two types of binding interactions involving the ‘So- matomedin B’ as well as the heparin-binding domain of VN a pl for direct association between both compo- nents! ’ The quantities of deposited PAI- into the growth substratum of endothelial cells strongly corre- lates with the concentration of ECM-associated VN. As a consequence, VN-containing focal adhesions associ- ated with PAL1 producing cells are protected against pericellular proteolysis.

The modification of these PAI- 1 -controlled processes as well as proteolytic modification of VN as PAI- co- factor and consequences for the cross-talk of vessel wall components was addressed in the present context. Acti- vation of the blood coagulation system finally leads to formation of the ternary VN-thrombin-antithrombin III complex which reaches vessel wall ECM by transcytosis through endothelial cells. Similar to multimeric VN (the predominant ECM-associated form of the adhesive pro-